Polycystic ovary syndrome (PCOS) is a leading cause of infertility in women. Five to 10% of premenopausal women are estimated to be affected by this disease, which is characterized by hyperandrogenemia, chronic anovulation, and is associated with other health risks such as type 2 diabetes mellitus and obesity. A primary defect in PCOS resides in the ovary, with increased (thecal/stromal) androgen production and follicular maturation arrest being the hallmarks. Unique changes in PCOS ovarian morphology, hyperplasia and thickening of the cortical ovarian stroma and hypertrophy of the stroma at the cortical-medullary boundary, suggest the possible involvement of the ovarian stroma. Previous investigators have postulated that the increased stromal density could be a cause of PCOS or a result of a primary ovarian deficit. We hypothesize that there is a bi-directional dialogue between the stroma and theca that influences the activities of these two compartments. The long-term objective of this project is to elucidate the role ovarian stromal cells play in the etiology of PCOS. This objective will be attained by completing the following specific aims: 1) define ovarian stromal cell paracrine interactions with thecal cells focusing on steroidogenesis and growth factor/growth factor binding protein production; and 2) determine whether PCOS ovarian stromal cells have functional defect(s) that result in the manifestation of the thecal PCOS phenotype, or whether stromal cells exhibit an altered phenotype because of interaction with PCOS thecal cells. To examine stromal cell function and paracrine theca interactions, cell culture experiments will be performed. The effects of androgens, progesterone, gonadotropins and cAMP treatment on stromal cell proliferation, steroidogenic potential and growth factor/growth factor binding protein production will be evaluated using radioimmunoassays, ELISAs, Western blots, and quantitative real-time RT-PCR. Paracrine interactions between stromal and thecal cells will be evaluated by testing conditioned medium from each cell type on the other cell type and by co-culture of thecal and stromal cells. Identification of the paracrine factor(s) expressed by ovarian stromal cells that influence theca cells will be initiated using standard fractionation procedures, followed by state-of-the-art proteomic and lipid/small molecule approaches. Lastly, we will examine PCOS stromal and thecal cell interactions, determining if they differ from normal ovarian stromal/thecal interactions. These studies should elucidate for the first time the functional role of ovarian stromal cells, and their role in PCOS. [unreadable] [unreadable]